期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 37, 期 1, 页码 180-186出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2004.05.007
关键词
human interleukin-4; refolding; heteronuclear NMR spectroscopy; cytokine
Interieukin-4 (IL4) is a multifunctional cytokine which plays a key role in the immune system. Several antagonists/agonists of IL4 are reported through mutagenesis studies, but their solution structural studies using nuclear magnetic resonance (NMR) spectroscopy are hindered as milligram quantities of isotopically labeled protein are required for structural refinements. In this work, a Histagged recombinant form of human IL4 was overexpressed in Escherichia coli under the control of a T7 promoter. The resulting inclusion bodies were separated from cellular debris by centrifugation and solubilized by 6 M guanidine-HCl in the presence of reducing agents. The denatured IL4 was immobilized on Ni2+ -fractogel beads and refolded in a single chromatographic step by gradual removal of denaturant. This protocol yielded 15-20 mg of isotope-enriched protein from 1 L of culture grown in minimal medium. The refolded protein was highly pure and was correctly folded as judged by its two-dimensional NMR spectrum. To show the successful application of this refolding protocol to IL4 variants, N-15-labeled Y124D-IL4 was also prepared and its first two-dimensional NMR spectrum was presented. (C) 2004 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据